Liposomes are completely closed lipid bilayer membranes which contain entrapped aqueous volume. Liposomes are vesicles which may be unilamellar (single membrane) or multilamellar (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. In the membrane bilayer, the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer, whereas the hydrophilic (polar) "heads" orient toward the aqueous phase. The basic structure of liposomes may be made by a variety of techniques known in the art.
The original liposome preparation of Bangham, et al. (J. Mol. Biol., 1965, 13:238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell" and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochim. Biophys. Acta., 1968, 135:624-638), and large unilamellar vesicles. Small unilamellar vesicles have a diameter of about 100 nm or less.
Unilamellar vesicles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT Application No. WO 86/,00238, published Jan. 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles" incorporated herein by reference. Vesicles made by this technique, called LUVETS, are extruded under pressure once or a number of times through a membrane filter. LUVETs will be understood to be included in the term "unilamellar vesicle".
Another class of multilamellar liposomes are those characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk, et al., monophasic vesicles as described in U.S. Pat. No. 4,588,578 to Fountain, et al. and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle. The FATMLV procedure is described in Bally et al., PCT Publication No. 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies", corresponding to U.S. Pat. No. 4,975,282, issued Dec. 4, 1990. U.S. Pat. No. 4,721,612to Janoff et al. describes steroidal liposomes for a variety of uses. The teachings of these references as to preparation and use of liposomes are incorporated herein by reference.
In the vaccine art immunogens are introduced into an organism in a manner so as to stimulate an immune response in the host organism. The induction of an immune response depends on many factors among which are believed to include the chemical composition and configuration of the immunogen, the immunogenic constitution of the challenged organism, and the manner and period of administration of the immunogen. An immune response has many facets, some of which are exhibited by the cells of the immune system, (e.g.,B-lymphocytes, T-lymphocytes, macrophages, and plasma cells). Immune system cells may participate in the immune response through interaction with immunogen, interaction with other cells of the immune system, the release of cytokines and reactivity to those cytokines. Immune response is conveniently (but arbitrarily) divided into two main categories--humoral and cell-mediated. The humoral component of the immune response includes production of immunoglobulins specific for the immunogen. The cell-mediated component includes the generation of delayed-type hypersensitivity and cytotoxic effector cells against the immunogen.
In some instances immune response is the result of an initial or priming dose of an immunogen that is followed by one or more booster exposures to the immunogen. Priming with relatively strong immunogens and liposomes is discussed in "Liposomal Enhancement of the Immunogenicity of Adenovirus Type 5 Hexon and Fiber Vaccines", Kramp, W. J. et al., Infection and Immunity, 25:771-773 (1979) and "Liposomes as Adjuvants with Immunopurified Tetanus Toxoid: the Immune Response", Davis, D. et al., Immunology Letters, 14:341-8 (1986/1987).
Ideally, an immunogen will exhibit two properties, the capacity to stimulate the formation of the corresponding antibodies and the propensity to react specifically with these antibodies. Immunogens bear one or more epitopes which are the smallest part of an immunogen recognizable by the combining site of an antibody or immunogloublin. In particular instances immunogens or fractions of immunogens or with particular presenting conditions the immune response precipitated by the desired immunogen is inadequate or nonexistent and insufficient immunity is produced. This is particularly the case with peptide or other small molecules used as immunogens.
In such cases the vaccine art recognizes the use of substances called adjuvants to potentiate an immune response when used in conjunction with an immunogen. Adjuvants are further used to elicit immune response sooner, or a greater response, or with less immunogen or to increase production of certain antibody subclasses that afford immunological protection, or to enhance components of the immune response (e.g., humoral, cellular).
Well known adjuvants are Freund's Adjuvants (and other oil emulsions), Bortedella Pertussis, aluminum salts (and other metal salts), Mycobacterial products (including muramyl dipeptides), and liposomes. As used herein the term "adjuvant" will be understood to mean a substance or material administered together or in conjunction with an immunogen which increases the immune response to that immunogen. Adjuvants may be in a number of forms including emulsion (e.g., Freund's adjuvant) gels (aluminum hydroxide gel) and particles (liposomes) or as a solid material.
It is believed that adjuvant activity can be effected by a number of factors. Among such factors are (a) carrier effect, (b) depot formation, (c) altered lymphocyte recirculation, (d) stimulation of T-lymphocytes, (e) direct stimulation of B-lymphocytes and (f) stimulation of macrophages.
With many adjuvants adverse reactions are seen. In some instances adverse reactions include granuloma formation at the site of injection, severe inflammation at the site of injection, pyrogenicity, adjuvant induced arthritis or other autoimmune response, or oncogenic response. Such reactions have hampered the use of adjuvants such as Freund's adjuvant.
In particular embodiments adjuvants are comprised of liposomes. U.S. Pat. No. 4,053,585 issued Oct. 17, 1977 to Allison et al. states that liposomes of a particular charge are adjuvants. Davis, D, et al., "Liposomes as Adjuvants with Immunopurified Tetanus Toxoid: Influence of Liposomal Characteristics", Immunology, 61:229-234 (1987) and; Gregoriadis, G. et al., "Liposomes as Immunological Adjuvants: Antigen Incorporation Studies", Vaccine, 5:145-151 (1987) report DMPC/cholesterol liposomes (1:1) and immunogen as giving minimally improved (over free immunogen) immunological response in unilamellar vesicles of a distinct dehydration/rehydration type with tetanus toxoid as the immunogen, a strong immunogen. In the Davis and in the Gregoriadis papers, the liposomal immunogenic response was only minimally distinguishable from the response of free immunogen. To distinguish the liposomal from free immunogen response it was necessary for the authors to dilute the tetanus toxoid to minimal response amounts.
Other substances such as immunomodulators (e.g., cytokines such as the interleukins) may be combined in adjuvants/vaccines as well. Humoral immune response may be measured by many well known methods. Single Radial Immunodifussion Assay (SRID), Enzyme Immunoassay (EIA) and Hemagglutination Inhibition Assay (HAI) are but a few of the commonly used assays of humoral immune response.
SRID utilizes a layer of a gel such as agarose containing the immunogen being tested. A well is cut in the gel and the serum being tested is placed in the well. Diffusion of the antibody out into the gel leads to the formation of a precipitin ring whose area is proportional to the concentration of the antibody in the serum being tested. EIA, also known as ELISA (Enzyme Linked Immunoassay), is used to determine total antibodies in a sample. The immunogen is adsorbed to the surface of a microtiter plate. The test serum is exposed to the plate followed by an enzyme linked immunogloublin, such as IgG. The enzyme activity adherent to the plate is quantified by any convenient means such as spectrophotometry and is proportional to the concentration of antibody directed against the immunogen present in the test sample.
HAI utilizes the capability of an immunogen such as viral proteins to agglutinate chicken red blood cells (or the like). The assay detects neutralizing antibodies, i.e. those antibodies able to inhibit hemagglutination. Dilutions of the test serum are incubated with a standard concentration of immunogen, followed by the addition of the red blood cells. The presence of neutralizing antibodies will inhibit the agglutination of the red blood cells by the immunogen.
Tests to measure cellular immune response include determination of delayed-type hypersensitivity or measuring the proliferative response of lymphocytes to target immunogen.
A variety of sterols and their water soluble derivatives have been used in cosmetics, pharmaceuticals and diagnostics. Of the water soluble sterols, for example, branched fatty acid cholesterol esters, steroid esters and PEG-phytosterols have been used in cosmetic preparations (U.S. Pat. No. 4,393,044 and European Patent Application No. 28,456; and Schrader, Drug and Cosmetic Industry, September, 1983, p. 33 and October 1983, p. 46). A number of water soluble cholesterols have been prepared and used as water-soluble standards for the determination of cholesterol levels in biological fluids (See, for example, U.S. Pat. Nos. 3,859,047 4,040,784; 4,042,330; 4,183,847; 4,189,400; and 4,224,229). Shinitzky et al. (1979, Proc. Natl. Acad. Sci. USA, 76, 5313) incubated tumor cells in tissue culture medium containing a low concentration of cholesterol and cholesteryl hemisuccinate. Incorporation of cholesterol or cholesteryl hemisuccinate into the cell membrane decreased membrane fluidity and increased membrane-lipid microviscosity.
Cholesterol and other sterols, have also been incorporated into phospholipid liposome membranes in order to alter the physical characteristics of the lipid bilayers. For example, Ellens, et al. (1984, Biophys. J. 45:70 abstract) discuss the effect of H+ on the stability of lipid vesicles composed of phosphatidylethanolamine and cholesteryl hemisuccinate. Brockerhoff and Ramsammy (1982, Biochim. Biophys. Acta. 691, 227) reported that bilayers can be constructed which consist entirely of cholesterol, provided that a stabilized hydrophilic anchor is provided. Multilamellar and unilamellar cholesterol liposomes have been prepared in a conventional manner. More recently, Janoff et al., U.S. Pat. No. 4,721,612, relevant portions of which are incorporated by reference herein, describe methods and compositions for the preparation of lipid vesicles, the bilayers of which comprise a salt form of an organic acid derivative of a sterol, for example, the tris-salt form of a sterol hemisuccinate. The vesicles of Janoff may entrap numerous bioactive agents including insulin, growth hormone, diazepam, indomethacin and tylosin, among others; however, most of the liposomes disclosed therein are not storage stable, i.e., capable of remaining stable for a period of at least two years under standard storage conditions.